Proteasome inhibition targets the KMT2A transcriptional complex in acute lymphoblastic leukemia

Rearrangments in Histone-lysine-N-methyltransferase 2A (KMT2Ar) are associated with pediatric, adult and therapy-induced acute leukemias. Infants with KMT2Ar acute lymphoblastic leukemia (ALL) have a poor prognosis with an event-free-survival of 38%. Herein we evaluate 1116 FDA approved compounds in primary KMT2Ar infant ALL specimens and identify a sensitivity to proteasome inhibition. Upon exposure to this class of agents, cells demonstrate a depletion of histone H2B monoubiquitination (H2Bub1) and histone H3 lysine 79 dimethylation (H3K79me2) at KMT2A target genes in addition to a downregulation of the KMT2A gene expression signature, providing evidence that it targets the KMT2A transcriptional complex and alters the epigenome. A cohort of relapsed/refractory KMT2Ar patients treated with this approach on a compassionate basis had an overall response rate of 90%. In conclusion, we report on a high throughput drug screen in primary pediatric leukemia specimens whose results translate into clinically meaningful responses. This innovative treatment approach is now being evaluated in a multi-institutional upfront trial for infants with newly diagnosed ALL.


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Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Experiments with patient samples: 6 patient samples were grown successfully in vitro and used for high throughput drug screening. Samples that did not grow in vitro were excluded. Histone Extractions and Analysis ( Figure 3A) -all six patient samples that demonstrated growth in vitro were included and four patient samples that did not grow in vitro were included in this experiment to ensure reproducibility across samples with varying growth characteristics. CUT&RUN PCR: Four patient samples that demonstrated growth in vitro for which there was sufficient specimens to meet the minimal input requirement were chosen. RNASeq ( Figure 3E)-5 patient samples that demonstrated growth in vitro were selected for this experiment. Samples were chosen based on sufficient material availability. Combinatorial drugging ( Figure 4B): Two patient samples that demonstrated growth in vitro and had sufficient material availability for combinatorial drugging were selected in addition to a cell line (SEM) that had the required characteristics (infant ALL with KMT2Ar).
no data exclusions Figure 1: Six passaged patient specimens were thawed and cultured in the presence of compounds at a single concentration of 10µM in duplicate Figure 3A: 10 unique patient samples were analyzed for H2Bub1 -3 samples in expt 1, 3 samples in expt 2, and 4 samples in expt 4. Figure 3B: 4 unique patient samples were analyzed by KMT2A CUT&RUN -2 samples in expt 1 and 2 samples in expt 2 Figure 3C: ChIP-RX was performed twice with similar results. A representative experiment is shown.  Figure 3E: Experiment was performed twice -the first time with two patient samples using gene expression arrays, the second time with five unique patient samples using RNA sequencing. Figure 3G: Four independent experiments Figure 4A: Two independent experiments Figure 4B: Three experiments with three unique samples (SEM cell line and two patient samples) Murine In Vivo Studies: Mice engrafted with leukemia were treated with DMSO, bortezomib, or bortezomib+vorinostat treatment. Mice were not randomized. Engraftment levels were not statistically different between cohorts. Patients treated with bortezomib and vorinostat containing regimens were not randomized, they received treatment on a compassionate use basis.
Blinding of murine in vivo studies and patients treated was not possible as administration of the correct drug was necessary. For murine experiments drug treatments were required to be specified on cage cards as per animal facility requirements.  Confirm that both raw and final processed data have been deposited in a public database such as GEO.

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Genome browser session Female NSG mice purchased from Jackson laboratories, (strain #005557) age 4 to 8 weeks. Mice were housed with a 12 hour light /